The natural products cyclosporin A, FK506, and rapamycin are potent immunosuppressants with realized or potential clinical applications in the prevention of graft rejection after organ transplantation and the treatment of autoimmune disorders (Borel, 1986; Kino et al., 1987; Martel et al., 1977). These drugs act by inhibiting intermediate steps in the signaling pathways that effect the T-cell response to antigen (for reviews see, Fruman et al., 1994; Kunz and Hall, 1993; Liu, 1993; Schreiber, 1991). This makes them useful probes for identifying the components of those pathways and determining their physiological roles.
The immunosuppressants interact with the immunophilins, which are small, soluble, receptor proteins that mediate their actions. Cyclosporin A (a cyclical undecapeptide) binds to cyclophilin A, whereas FK506 and rapamycin (two related macrolide antibiotics) bind to a distinct receptor protein, FKBP12 (Handschumacher et al., 1984; Harding et al., 1989; Siekierka et al., 1989). Though cyclophilin and FKBP12 differ markedly in amino acid sequence, both immunophilins have peplidyl-prolyl cis-trans isomerization (rotamase) activity, which is inhibited by their respective ligands (for review, see Heitman et al., 1992). However, this inhibition does not appear to explain the effects of the immunosuppressants (Bierer et al., 1990a, b; Tropschug et al., 1989). Instead, the action of cyclosporin A and FK506 derives from the binding of the drug-receptor complexes to the calcium-activated protein phosphatase, calcineurin (Liu et al., 1991). This association inhibits the catalytic activity of the phosphatase, which is required for the Ca++-dependent initial step in the activation of the T-lymphocyte via the T-cell receptor (Flanagan et al., 1991; Kronke et al., 1984).
On the other hand, rapamycin appears to block a later, Ca++-independent stage in the T-cell response. This drug selectively inhibits the IL-2 stimulated G1 to S cell-cycle transition that initiates T-cell proliferation (Dumont et al., 1990b). Although this inhibition has been correlated with the decreased activity of the 70 kDa S6 kinase (pp70S6K), a known downstream effector of the IL-2 receptor, the FKBP12-rapamycin complex does not appear to interact directly with this kinase (Chung et al., 1992; Kuo et al., 1992). Similarly, in T-cells and other cell types, rapamycin blocks progression of the cell cycle by preventing the activation of the cyclin-dependent kinases p33cdk2 and p34cdc2, but an association of the drug-immunophilin complex with the kinases or their respective cyclins has not been demonstrated (Albers et al., 1993; Jayaraman and Marks, 1993; Morice et al., 1993).
In the budding yeast S. cerevisiae, rapamycin also causes an arrest in the G1 phase of the cell cycle through its binding to a highly conserved FKBP12 homologue (Heitman et al., 1991b). The toxicity of the drug for yeast cells has allowed, through genetic selection, the identification of two homologous genes, which, when mutated, render the cells rapamycin-resistant (Heitman et al., 1991a). This led to the proposal that the products of these genes, which show some amino acid homology to the catalytic domain of the p110 subunit of PI-3 kinase, are the Targets Of Rapamycin and hence to the designation of the genes as TOR1 and TOR2 (Kunz et al., 1993). Direct support for this proposal, however, has not been presented and how the TOR gene products confer sensitivity to rapamycin remains to be elucidated. Alternatively, it has been suggested that in the signaling pathway blocked by rapamycin, the TOR proteins, like the S6 kinase and the cyclin-dependent kinases, lie downstream from the direct target of the FKBP12-rapamycin complex (Albers et al., 1993; Helliwell et al., 1994). This model assumes that the TOR mutations lead to the constitutive activation of the TOR1 and TOR2 proteins.
Besides binding to calcineurin in a FK506-dependent manner, FKBP12 can also interact with calcium-channel proteins, the ryanodine receptor, which mediates calcium induced calcium released (Jayaraman et al., 1992; Timerman et al., 1993) and the inositol 1,4,5,-triphosphate (IP3) receptor (A. Cameron, A. Kaplin, D. Sabatini, J. Steiner, S. Snyder, unpublished). These associations do not require FK506 or rapamycin; indeed these drugs dissociate the FKBP12-channel complex.
There is a need in the art to identify, isolate, and purify the mammalian cellular proteins that interact with FKBP12 only in the presence of rapamycin. Such proteins play a role in immunological, neurological, and cell cycle functions.
It is an object of the invention to provide isolated, purified cDNA molecules encoding rapamycin and FKBP target molecules.
It is another object of the invention to provide fusion proteins comprising rapamycin and FKBP targets.
It is still another object of the invention to provide an isolated and purified rapamycin and FKBP target molecule.
It is still another object of the invention to provide an expression construct which directs synthesis in a cell of an RNA molecule which inibits expression of a rapamycin and FKBP target molecule.
It is yet another object of the invention to provide isolated, purified cDNA molecules which are complementary to genes encoding rapamcyin and FKBP target molecules.
It is an object of the invention to provide a method of screening for potential therapeutic agents.
It is another object of the invention to provide a method of purifying a rapamycin and FKBP target molecule.
It is still another object of the invention to provide a method of isolating DNA sequences which code for rapamycin and FKBP target molecules.
These and other objects of the inveniton are provided by one or more of the embodiments described below. In one embodiment of the invention an isolated, purified cDNA molecule is provided which encodes RAFT1, a protein having the amino acid sequence shown in SEQ ID NO:1.
In another embodiment of the invention a fusion protein comprising the amino acid sequence shown in SEQ ID NO:1, is provided.
In yet another embodiment of the invention an isolated and purified RAFT1 protein having the amino acid sequence shown in SEQ ID NO:1 is provided. Also provided is an isolated and purified RAFT2 protein, having an apparent molecular weight on SDS polyacrylamide gels of 35 kDa. Also provided is an isolated and purified mammalian RAFT protein which is free of proteins which do not bind to rapamycin and FKBP12. Also provided is a mammalian RAFT protein prepared by the process of:
contacting a preparation of mammalian proteins with FKBP12 in the presence of rapamycin;
isolating mammalian proteins which bind to FKBP12 in the presence of rapamycin from those mammalian proteins which do not bind; and
dissociating bound mammalian proteins from FKBP12 to provide a mammalian RAFT protein.
In still another embodiment of the invention an expression construct is provided. The expression construct comprises a promoter operably linked to at least 20 nucleotides of the antisense strand of RAFT1 cDNA, said expression construct directing synthesis in a cell of an RNA molecule which is complementary to RAFT1 RNA.
In another embodiment of the invention an isolated, purified cDNA molecule comprising at least 20 nucleotides of the sequence of RAFT1 is provided.
In yet another embodiment of the invention a method of screening substances for potential as therapeutic agents is provided. The method comprises the steps of:
contacting a substance to be tested with three components: (a) FKBP12, (b) rapamycin, and (c) a protein selected from the group consisting of RAFT1 and RAFT2;
determining the amount of one of said components bound to the other components in the presence and absence of said substance; a substance which increases or decreases the amount of said component bound being a potential therapeutic agent.
In one embodiment of the invention a method of purifying a mammalian RAFT protein is provided. The method comprises the steps of:
contacting a preparation of mammalian proteins with FKBP12 in the presence of rapamycin;
isolating mammalian proteins which bind to FKBP12 in the presence of rapamycin from those mammalian proteins which do not bind;
dissociating bound mammalian proteins from FKBP12 to provide a mammalian RAFT protein.
In another embodiment of the invention methods of isolating mammalian RAFT DNA sequences are provided. One of the methods comprises:
probing a library of mammalian DNA sequences with a probe which comprises at least 15 contiguous nucleotides selected from the sequence of RAFT1 cDNA.
Another of the methods comprises:
amplifying a DNA sequence using at least one primer which comprises at least 10 contiguous nucleotides selected from the sequence of RAFT1 cDNA.
These and other embodiments of the invention provide the art with potent tools for identifying drugs useful in the treatment of immunological, neurological, and cell cycle-related diseases and defects.